1. Field of the Invention
The present invention relates generally to the field of immunology. More specifically, the present invention involves identification of dominant CD8 T cell epitope in the Human Papillomavirus (HPV) proteins and its use in treating cancer such as cervical cancer.
2. Description of the Related Art
Cervical cancer is the second most common malignancy among women worldwide (World Health Organization, 1990) with 400,000 new cases being diagnosed annually (Parkin, D. et al., 1999). Annually 12,000 to 14,000 new cases of squamous cell cancer of the cervix are reported in the United States (Silverberg, E. and Lubera, J., 1988), resulting in about 3,500 deaths per year. The link between human papilloma virus (HPV) and the development of cervical cancer is well known. Among the over one hundred different types of HPV, at least 15 are strongly associated with invasive squamous cell cancer of the cervix (Munoz, N. et al., 2003). HPV16 is the one most commonly found associated with this cancer (Beaudenon et al., 1986; Crum et al., 1985).
HPV infection is also associated with the precursor lesion of cervical cancer, squamous intraepithelial lesion (SIL) (Beaudenon, et al., 1986; Crum et al., 1985; Reid, R., 1987; Lorincz et al., 1986; Lorincz et al., 1987; Fuchs, et al., 1988). While most low-grade squamous intraepithelial lesions prospectively regress spontaneously (Koutsky et al., 1992; Richart and Barron, 1969), some progress to high-grade squamous intraepithelial lesions. These high-grade lesions, in particular, cervical intraepithelial neoplasia 3 (CIN-3) are associated with a high rate progression to invasive cervical cancer (Nash et al., 1987; Campion et al., 1986).
Transformation to a malignant phenotype by HPV is mediated by two early gene products, E6 and E7. Both of these viral proteins have been shown to interact with the products of cellular human tumor suppressor genes. The E6 protein can bind and promote degradation of cell-encoded p53, while the E7 protein interacts with the retinoblastoma susceptibility gene product (Crook et al., 1991; Heck et al., 1992; Scheffner et al., 1990). The expression of E6 and E7 open reading frames has been shown to be necessary and sufficient for transformation of human cells by HPV 16 (Schlegel, R. et al., 1988; Storey, A. et al., 1988; Pirisi, L. et al., 1987). Therefore, the E6 and E7 proteins can serve as potential targets when developing new preventative and therapeutic modalities.
Cell-mediated immunity has been shown to play an important role in controlling HPV infection and HPV-associated diseases. CD8-positive, MHC class I-restricted cytotoxic T lymphocytes (CTLs) are known to be responsible for recognizing and killing virus-infected host cells and virus-induced tumors (Greenberg, P. D., 1991). Immunohistochemical analyses of squamous intraepithelial lesions and cervical cancer specimens have demonstrated the presence of activated CTLs in lesions (Bontkes, H. J. et al., 1997). Studies using mouse models have demonstrated that immunization with HPV 16 E6 or E7-transfected non-tumorigenic fibroblasts can lead to regression of tumors expressing E6 or E7 respectively, and that these events are mediated by CD8-positive CTLs (Chen, L. P. et al., 1991; Chen, L. et al., 1992).
In humans, HPV16 E6 and/or E7-specific CTLs have been identified in women with cervical cancer and women with squamous intraepithelial lesions. One group stimulated the peripheral blood mononuclear cells (PBMCs) from cervical cancer patients with an HLA-A2-restricted HPV16 E7 peptide (E7 11-20, SEQ ID NO: 3) and demonstrated that CTLs were capable of lysing HLA-matched HPV16 E7 11-20 (SEQ ID NO: 3)-pulsed targets in two of three patients (Alexander et al., 1996). Another group identified HPV-specific CTLs in lymph nodes and tumors of cervical cancer patients (Evans et al., 1997). In some patients with squamous intraepithelial lesions, CTLs to HPV16 E6 and E7 were demonstrated in PBMCs stimulated in vitro with the cervical carcinoma line CaSki (Evans et al., 1996).
HPV16 E6 and E7-specific CTLs have also been demonstrated in subjects who had evidence of HPV16 infection but who had not developed squamous intraepithelial lesions. In a small cross-sectional study, the percentage of subjects who demonstrated HPV16 E6 and/or E7-specific CTLs was higher in a group of women with HPV16 infection who had not developed squamous intraepithelial lesions, compared to a group of women with HPV16 infection who had developed squamous intraepithelial lesions. The effector cell phenotypes in these women who had not developed squamous intraepithelial lesions were shown to be CD4- and CD8-positive T lymphocytes. In women with PCR-detected cervical HPV16 infection, the association between HPV16 E6 and E7-specific CTLs and HPV16 persistence was examined using a longitudinal study design involving multiple CTL assays (Nakagawa, M. et al., 2002). Lack of CTL response to the HPV16 E6 protein but not the E7 protein was correlated with persistent HPV16 infection, suggesting that CTL responses to E6 and E7 are likely to be important at different stages during the course of infection. These studies suggested that the development of cervical cancer may due to insufficient cell-mediated immunity to HPV and that one of the possible modalities for treatment of cervical cancer may be enhancing such response as would be done in dendritic cell immunotherapy.
CD8-positive CTL recognize foreign peptides that are 8 to 11 amino acids in length and bound to and presented by HLA class 1 molecules. These peptides are called T cell epitopes. Both mouse (Ressing, M. E. et al., 1995; Sadovnikova, E. et al., 1994) and human (Ressing et al., 1995: Tarpey, I. et al., 1994) systems have been used to identify the antigenic epitopes of HPV. One group identified the potential CTL epitopes of HPV16 E6 and E7 proteins for five common HLA types by measuring binding of each of the 150 nonamer peptides using purified HLA molecules and radlolabeled peptides (Kest, W. M. et al., 1994). The immunogenicity of 9 of these potential antigenic epitopes for HLA-2.1 was tested (Ressing, M. E. et al., 1995). In vivo, 4 immunogenic peptides were identified (E6 29-38 (SEQ ID NO: 1), E7 11-20 (SEQ ID NO: 3), E7 (82-90) and E7 (86-93)) using HLA-2.1 transgenic mice. Additionally, in vitro CTL induction of human PBMCs confirmed the immunogenicity of 3 of the 4 peptides (E7 11-20 (SEQ ID NO: 3), E7 (82-90) and E7 (86-93)). CTLs to one of these peptides, E7 11-20 (SEQ ID NO: 3) have been demonstrated in patients with SIL and in cancer patients. However, since responses to this peptide were found in cancer patients, it is not clear whether this peptide played a protective role.
Another study identified antigenic epitopes of HPV16 E6 and E7 proteins by using overlapping peptides of these proteins to stimulate PBMCs from a healthy donor and binding assays to find candidate epitopes (Bourgault Villada, I. et al., 2000). This approach enabled the Identification of HLA-B18 epitopes, E6 80-88 (ISEYRHYCY; SEQ ID NO: 2) and E7 44-52 (QAEPDRAHY; SEQ ID NO: 4). It was also shown that E6 (80-88) was a naturally processed epitope that could be recognized by T cells from a patient with HSIL. Although the binding of the peptide to the HLA molecule was demonstrated, the strength of the T cell response to these antigenic epitopes compared with other T cell epitopes was not assessed. Since response to E6 80-88 (SEQ ID NO: 2) epitope was demonstrated in patient who had developed high-grade SIL, it was not clear whether this peptide had a protective effect.
Additionally, some work has also been carried out to identify antigenic epitopes of another common high-risk type, HPV 18. Using the same approach as was taken for HPV16, HLA-A2.1 binding synthetic peptides of HPV18 E6 protein were identified (Yoon, H. et al., 1998). Some of these binding peptides were also shown to be antigenic by demonstrating in vitro cytotoxicity (Table 1). An HLA-Cw4-restricted HPV18 L1 epitope, NVFPIFLQM (SEQ ID NO: 14) was identified by eluting and sequencing peptides from purified class I MHC molecules of a cervical cell line (Garcia, A. M. at al., 1999).
Table 1 lists a small number of HPV CD8 T lymphocyte epitopes shown to be antigenic in human experimental systems by demonstrating peptide-specific cytotoxicity. Except for the HLA-B18-restricted epitopes identified by Bourgault Villada et al., all were pre-selected for the given HLA types. None of the antigenic epitopes were identified based on the magnitude of T cell response regardless of the restricting HLA molecules.
TABLE 1High-risk HPV peptide antigens for CD8 T lymphocytesshown to be antigenic in human experimental systems bydemonstrating peptide-specific cytotoxicity or γ-IFN secretion.HPVHLATYPEPEPTIDETYPESEQUENCESEQ. ID. NO.HPV16E6 29-38A2.1TIHDIILECV1E6 80-88B18ISEYRHYCY2E7 11-20A2.1YMLDLQPETT3E7 44-52B18QAEPDRAHY4E7 82-90A2.1LLMGTLGIV5E7 86-93A2.1TLGIVCPI6L1 323-331A2.1ICWGNQLFV7HPV18E6 24-33A2.1SLQDIEITCV8E6 25-33A2.1LQDIEITCV9E6 40-48A2.1ELTEVFEFA10E6 47-55A2.1FAFKDLFVV11E6 92-101A2.1KLTNTGLYNL12E6 13-21A2.1KLPDLCTEL13L1 54-62Cw4NVFPIFLQM14
Dendritic cells are the most potent antigen-presenting cells and are capable of sensitizing T cells to new and recall antigens (Fong, L. et al., 2001). Recent advances in the knowledge of dendritic cell differentiation steps and in the technical abilities to prepare them in a large quantity enabled development of immunotherapy protocols for the treatment of variety of cancers. The results of clinical trials for different types of cancers have been reported but the best-studied tumors are malignant melanoma, prostate cancer, colorectal carcinoma and multiple myeloma (Ridgway, D. et al., 2003). The results of a small clinical trial of cervical cancer patients have been published in which autologous dendritic cells were pulsed with HPV16 E6 or HPV1 8 E7 protein (Ferrera et al., 2003). Although the therapy was well-tolerated and there was evidence of humoral and cell-mediated immune responses in some patients, no objective clinical response was reported.
Thus, prior art is deficient in peptide antigens derived from the HPV16 E6 and E7 proteins that have been identified based on the magnitude of T cell responses to be used as sources of antigens for dendritic cell immunotherapy for cervical cancer. The present invention fulfills this long-standing need and desire in the art.